Flavin-dependent oxidases having cannabinoid synthase activity

Abstract

The disclosure relates to a non-natural flavin-dependent oxidase comprising at least one amino acid variation as compared to a wild type flavin-dependent oxidase, wherein the non-natural flavin-dependent oxidase does not comprise a disulfide bond, and wherein the non-natural flavin-dependent oxidase is capable of oxidative cyclization of a prenylated aromatic compound into a cannabinoid. The disclosure also relates to a nucleic acid, an expression construct, and an engineered cell for making the non-natural flavin-dependent oxidase. Also provided are compositions comprising the non-natural flavin-dependent oxidase; isolated non-natural flavin-dependent oxidase and methods of making the same; cell extracts comprising the non-natural flavin-dependent oxidase; methods of making cannabinoids; and compositions comprising a cannabinoid and/or the non-natural flavin-dependent oxidase.

1 . A non-natural flavin-dependent oxidase comprising at least one amino acid variation as compared to a wild type flavin-dependent oxidase, wherein the non-natural flavin-dependent oxidase: (i) does not comprise a disulfide bond (ii) is capable of oxidative cyclization of a prenylated aromatic compound into a cannabinoid, and (iii) comprises at least 70% sequence identity to any one of SEQ ID NOs:1-3, any one of SEQ ID NOs:10-13, or any one of SEQ ID NOs:17-19. 
     
     
         2 . The non-natural flavin-dependent oxidase of  claim 1 , wherein the prenylated aromatic compound is cannabigerolic acid (CBGA), cannabigerorcinic acid (CBGOA), cannabigerivarinic acid (CBGVA), cannabigerorcinol (CBGO), cannabigerivarinol (CBGV), or cannabigerol (CBG). 
     
     
         3 . The non-natural flavin-dependent oxidase of  claim 1 , wherein the non-natural flavin-dependent oxidase is not glycosylated. 
     
     
         4 . The non-natural flavin-dependent oxidase of  claim 1 , wherein the non-natural flavin-dependent oxidase comprises a monovalently bound FAD cofactor, or wherein the non-natural flavin-dependent oxidase comprises a bivalently bound FAD cofactor. 
     
     
         5 . The non-natural flavin-dependent oxidase of  claim 1 , wherein the non-natural flavin-dependent oxidase is capable of oxidative cyclization of a prenylated aromatic compound into a cannabinoid at about pH 7.5. 
     
     
         6 . The non-natural flavin-dependent oxidase of  claim 1 , wherein catalytic activity of the non-natural flavin-dependent oxidase is substantially the same from about pH 5 to about pH 8. 
     
     
         7 . The non-natural flavin-dependent oxidase of  claim 1 , comprising at least 80% sequence identity to any one of SEQ ID NOs:1-3, any one of SEQ ID NOs:10-13, or any one of SEQ ID NOs:17-19, optionally comprising at least 90% sequence identity to any one of SEQ ID NOs:1-3, any one of SEQ ID NOs:10-13, or any one of SEQ ID NOs:17-19. 
     
     
         8 . The non-natural flavin-dependent oxidase of  claim 7 , comprising at least 90% sequence identity to any one of SEQ ID NOs:1-3, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:19. 
     
     
         9 . The non-natural flavin-dependent oxidase of  claim 6 , wherein the non-natural flavin-dependent oxidase converts CBGA to cannabichromenic acid (CBCA), cannabidiolic acid (CBDA), and/or Δ9-tetrahydrocannabinolic acid (THCA), optionally wherein the converting is at about pH 4 to about pH 9. 
     
     
         10 . The non-natural flavin-dependent oxidase of  claim 1 , further comprising an affinity tag, a purification tag, a solubility tag, or a combination thereof. 
     
     
         11 . A polynucleotide comprising a nucleic acid sequence encoding the non-natural flavin-dependent oxidase of  claim 1 . 
     
     
         12 . A polynucleotide comprising: (a) a nucleic acid sequence encoding a polypeptide comprising at least 80% sequence identity to any one of SEQ ID NOs:1-3, any one of SEQ ID NOs:10-13, or any one of SEQ ID NOs:17-19; and (b) a heterologous regulatory element operably linked to the nucleic acid sequence. 
     
     
         13 . The polynucleotide of  claim 12 , wherein the polypeptide comprises at least 90% sequence identity to any one of SEQ ID NOs:1-3, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:19. 
     
     
         14 . An expression construct comprising the polynucleotide of  claim 11 . 
     
     
         15 . An engineered cell comprising the non-natural flavin-dependent oxidase of  claim 1 . 
     
     
         16 . The engineered cell of  claim 15 , further comprising a cannabinoid biosynthesis pathway enzyme. 
     
     
         17 . The engineered cell of  claim 16 , wherein the cannabinoid biosynthesis pathway enzyme comprises olivetol synthase (OLS), olivetolic acid cyclase (OAC), prenyltransferase, a geranyl pyrophosphate (GPP) biosynthesis pathway enzyme, or a combination thereof. 
     
     
         18 . The engineered cell of  claim 15 , wherein the cell is a bacterial cell, optionally wherein the cell is an  Escherichia coli  cell. 
     
     
         19 . A cell extract or cell culture medium comprising cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabigerol (CBG), cannabichromene (CBC), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabigerorcinic acid (CBGOA), cannabiorcichromenic acid (CBCOA), cannabidiorcinic acid (CBDOA), tetrahydrocannabiorcinic acid (THCOA), cannabigerivarinic acid (CBGVA), cannabichromevarinic acid (CBCVA), cannabidivarinic acid (CBDVA), tetrahydrocannabivarinic acid (THCVA), cannabigerorcinol (CBGO), cannabichromeorcin (CBCO), cannabidiorcin (CBDO), tetrahydrocannabiorcin (THCO), cannabigerivarinol (CBGV), cannabichromevarin (CBCV), cannabidivarin (CBDV), tetrahydrocannabivarin (THCV), an isomer, analog or derivative thereof, or a combination thereof derived from the engineered cell of  claim 15 . 
     
     
         20 . A method of making a cannabinoid selected from CBCA, CBC, CBCOA, CBCVA, CBCO, CBCV, CBDA, CBD, CBDOA, CBDVA, CBDO, CBDV, THCA, THC, THCOA, THCVA, THCO, THCV, an isomer, analog or derivative thereof, or a combination thereof, comprising: culturing the engineered cell of  claim 15 . 
     
     
         21 - 32 . (canceled)